This invention relates to plasminogen activators which are useful thrombolytic agents. More particularly, this invention relates to the large scale production of plasminogen activators from cultured normal human colon cells.
It is known that plasminogen activators (PA) are widely distributed throughout the body and can be purified from tissue extracts. Typical examples of tissue sources are kidney and lung tissues. The best characterized of these plasminogen activators fall into two major groups, urokinase-type plasminogen activator (u-PA) and tissue-type plasminogen activator (t-PA). u-PA and t-PA are present in ng/ml concentrations in human plasma but are immunologically unrelated. t-PA has been demonstrated to have higher affinity for fibrin than u-PA. u-PA products isolated and purified from human urine and from mammalian kidney cells are pharmaceutically available as thrombolytic agents.
Due to the extremely low concentration of t-PA in blood and tissue extracts, other sources and means of producing this preferred thrombolytic agent have been sought after.
One method of producing t-PA on a large scale comprises isolating the protein from the culture fluid of human melanoma cells grown under in vitro cell culture conditions. An established human melanoma cell line (Bowes) has been used for this purpose. See, for example, European Patent Application 41,766, published Dec. 16, 1981; Rijken and Collen, J. Biol. Chem. 256(13), 7035-7041 (1981); and Kluft et al., Adv. Biotech. Proc. 2, Alan R. Liss, Inc., 1983, pp. 97-110. Genetic information from this cell line also has been embodied in E. coli by conventional recombinant DNA gene splicing methods to permit the production of the t-PA protein by that microorganism. See, for example, UK Patent Application 2,119,804, published Nov. 23, 1983; Pennica et al., Nature 301, 214-221 (1983); and Vehar et al., Biotech. 2(12), 1051-1057 (1984). Such t-PA material from Bowes melanoma has been administered to humans with some measure of effectiveness. See Collen et al., Circulation 70 16, 1012-1017 (1984).
Notwithstanding the apparent utility of the t-PA derived from Bowes melanoma, the use of cancer cells or genetic information derived from cancer cells can raise uncertain drug regulatory problems in the therapeutic use of such materials. Thus, it is known that cancer cells (transformed cells) can produce human transforming growth factors. See, for example, Delarco and Todaro, Proc. Natl. Acad. Sci. USA 75, 4001-4005 (1978), and Todaro et al., Ibid., 77, 5258-5262 (1980). Even the smallest amount of residual DNA from the cancer cells can be integrated into and expressed in the E. coli or genetically engineered mammalian cells, thereby raising the possibility of harmful effects if t-PA from such source is administered to the patient. Although the risks may be small by judicious use of various purification techniques and appropriate monitoring of patients, it still would be preferable to use a t-PA that was not derived from cancer cells either directly or indirectly. The possible presence of viral genetic material or oncogene product can raise significant objections to the use of clinical material thus derived from transformed cells.
Accordingly, the production of tissue-type plasminogen activators from normal human cells on a large scale would be highly desirable. Cultured normal human cells have been used as a source of t-PA as can be seen from U.S. Pat. Nos. 4,505,893 and 4,550,080. Although various cell sources are mentioned in said patent, apparently only primary embryonic (or fetal) kidney, lung, foreskin, skin and small intestines (Flow Laboratories) were actually cultured according to the disclosure. Brouty-Boye et al., Biotech. 2(12), 1058-1062 (1984), also disclose the use of normal human embryonic lung cells for the production of t-PA. Rijken and Collen, J. Biol. Chem. 256 (13), 7035-7041 (1981), and Pohl et al., FEBS Lett. 168(1), 29-32 (1984), disclose the use of human uterine tissue as a t-PA source material.